The overall objective of this proposal is to develop methods for the cost-effective, large-scale synthesis (up to kilograms amounts) of capped RNA. Companies such as Wyeth Vaccines, AlphaVax and Merix Biosciences are using capped RNA to produce human vaccines against cancers and infectious diseases such as HIV and biowarfare agents. The mMESSAGEmMACHINE(r) kit, based on Ambion's patented technology, provides the customer with high-yielding transcription reactions for the synthesis of capped RNA. Although this technology is the best available on the market for producing capped RNA, the cost of making capped RNA using this technology under cGMP conditions may be too cost prohibitive to make these RNA-based vaccine strategies commercially feasible. Additionally, the current technology produces a mixture of uncapped, correctly capped and "reverse" capped RNA. Our goals are to decrease the cost of the capped RNA required per human subject by at least 100-fold, to increase the purity of the capped RNA such that >90% of the RNA is correctly capped and to develop methods for assessing the purity of the final RNA product. By developing a new reaction system, we could theoretically increase the yield of these reactions by as much a 8-fold. Preliminary experiments increased the yield up to 3-fold based on this novel strategy. By increasing the ratio of the cap analog to GTP concentrations in these reaction systems, we should be able to increase the percentage of capped RNA in a reaction to 90% and further, by using a novel "anti-reverse cap analog" we will incorporate all of the cap in the correct, functional orientation. Increasing the resistance of the capped RNA to nucleases is one strategy to decrease the amount of RNA required per human subject and we will test various strategies for increasing the stability of the capped RNA in a cell while maintaining its translational competency. Finally, not all of the transcription components required for generating maximum yields are available as an FDA approved component. These proteins will be cloned and expressed in systems approved by the FDA.